Selective dendrimer delivery to brain tumors

ABSTRACT

A composition comprising poly(amidoamine) (PAMAM) hydroxyl-terminated dendrimers covalently linked to at least one therapeutic, prophylactic or diagnostic agent for the treatment or alleviation of one or more symptoms of a brain tumor have been developed. The dendrimers comprise one or more ethylene diamine-core poly(amidoamine) (PAMAM) hydroxyl-terminated generation-4, 5, 6, 7, 8, 9, or 10, most preferably generation 6 (G4-10-OH) dendrimers. The G6 dendrimers have demonstrated unexpectedly high uptake into the brain. The dendrimers provide a means for selective delivery through the blood brain barrier (“BBB”) of chemotherapeutic, immunotherapeutic and palliative agents. The dendrimers also have the advantage that two different classes of compounds, having one or more mechanisms of action can be bound to the dendrimers, providing simultaneous delivery. The dendrimers may be administered alone by intravenous injection, or as part of a multi-prong therapy with radiation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/502,739, filed Feb. 8, 2017, which is a National Phase applicationunder 35 U.S.C. § 371 of PCT/US2015/045104, filed Aug. 13, 2015, whichclaims priority to and benefit of U.S. Provisional Patent ApplicationNos. 62/036,675 filed Aug. 13, 2014, 62/036,839, filed Aug. 13, 2014,and 62/059,240 filed Oct. 3, 2014, which are incorporated hereby byreference in their entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted as a text file named“JHU_C_13214_PCT_substitute_ST25.txt,” created on Mar. 12, 2018, andhaving a size of 3,192 bytes is hereby incorporated by referencepursuant to 37 C.F.R. § 1.52(e)(5).

FIELD OF THE INVENTION

The present invention is generally in the field of delivery ofchemotherapeutic, immunotherapeutic and palliative drugs to the brainfor treatment of brain tumors and associated symptoms.

BACKGROUND OF THE INVENTION

A brain tumor is an abnormal growth of tissue in the brain or centralspine that can disrupt proper brain function. Doctors refer to a tumorbased on where the tumor cells originated, and whether they arecancerous (malignant) or not (benign). The least aggressive type ofbrain tumor is often called a benign brain tumor. They originate fromcells within or surrounding the brain, do not contain cancer cells, growslowly, and typically have clear borders that do not spread into othertissue. Malignant brain tumors contain cancer cells and often do nothave clear borders. They are considered to be life threatening becausethey grow rapidly and invade surrounding brain tissue. Tumors that startin cells of the brain are called primary brain tumors. Primary braintumors may spread to other parts of the brain or to the spine, butrarely to other organs. Metastatic or secondary brain tumors begin inanother part of the body and then spread to the brain. These tumors aremore common than primary brain tumors and are named by the location inwhich they begin.

There are over 120 types of brain and central nervous system tumors.Brain and spinal cord tumors are different for everyone. They form indifferent areas, develop from different cell types, and may havedifferent treatment options. Often, low-grade tumors (grade I and II),which are not aggressive, are treated with watchful monitoring orsurgery alone. Though all tumors are monitored with repeat scans, gradeII tumors are watched more closely after surgery and over time to makesure there is no recurrence. Higher grade tumors (grade III and IV),which are malignant and can grow quickly, are more difficult to removeand require additional treatments beyond surgery, such as radiation orchemotherapy. Microscopic tumor cells can remain after surgery and willeventually grow back. All treatments, therefore, are intended to prolongand improve life for as long as possible.

For a low-grade brain tumor, surgery may be the only treatment neededespecially if all of the tumor can be removed. If there is visible tumorremaining after surgery, radiation and chemotherapy may be used. Forhigher-grade tumors, treatment usually begins with surgery, followed byradiation therapy and chemotherapy. Additional treatment options forhigh-grade tumors include X-rays and other forms of radiation to destroytumor cells or delay tumor growth; chemotherapy to kill rapidly dividingcells; targeted therapy which focuses on a specific element of a cell,such as molecules or pathways required for cell growth, in order to usethem as a target; and locally or regionally delivered treatment thatproduces electric fields to disrupt the rapid cell division exhibited bycancer cells by creating alternating, “wave-like” electric fields thattravel across their region of usage in different directions.

Successfully treating brain tumors can be challenging. The body'sblood-brain barrier normally protects the brain and spinal cord fromharmful chemicals entering those structures through the bloodstream.However, this barrier also keeps out many types of chemotherapy. Surgerycan be difficult if the tumor is near a delicate part of the brain orspinal cord. Even when the surgeon can completely remove the originaltumor, there may be parts of the tumor remaining that are too small tobe seen or removed during surgery. Radiation therapy can damage healthytissue.

A brain tumor and its treatment often cause side effects. In addition totreatment to slow, stop, or eliminate the tumor, an important part ofcare is relieving a person's symptoms and side effects. This approach iscalled palliative or supportive care, and it includes supporting thepatient with his or her physical, emotional, and social needs. Painmedication to help manage the pain from headaches, a common symptom of abrain tumor. Often, corticosteroids are used to lower swelling in thebrain, which can lessen pain from the swelling without the need forprescription pain medications. Antiseizure medication is used to helpcontrol seizures.

Surgery is commonly used to remove all or part of brain tumors.Sometimes, surgery cannot be performed because the tumor is located in aplace the surgeon cannot reach or is near a vital structure; thesetumors are called inoperable.

The goal of chemotherapy can be to destroy cancer cells remaining aftersurgery, slow a tumor's growth, or reduce symptoms. A chemotherapyregimen usually consists of a specific number of cycles given over a setperiod of time. A patient may receive one drug at a time or combinationsof different drugs at the same time. Common ways to give chemotherapyinclude a pill or capsule that is swallowed (orally) or by intravenous(IV). Some drugs are better at going through the blood-brain barrier,and these drugs are often used for a brain tumor because of thisability. Gliadel wafers are one way to give the drug carmustine, whichinvolves placing the wafers in the area where the tumor was removedduring surgery. For people with glioblastoma, the latest standard ofcare is radiation therapy with daily low-dose temozolomide (Temodar),followed by monthly doses of temozolomide after radiation therapy forsix months to one year. A combination of three drugs, lomustine (CeeNU),procarbazine (Matulane), and vincristine (Vincasar) have been used alongwith radiation therapy. This approach has helped lengthen the lives ofpatients with grade III oligodendroglioma with a 1p19q co-deletion whengiven either before or right after radiation therapy. It has also beenshown to lengthen lives of patients when given after radiation therapyfor low-grade tumors that could not be completely removed with surgery.The side effects of chemotherapy depend on the individual and the doseused, but they can include fatigue, risk of infection, nausea andvomiting, hair loss, loss of appetite and diarrhea. These side effectsusually go away once treatment is finished. Rarely, certain drugs maycause some hearing loss. Others may cause kidney damage. Patients may begiven extra fluid by IV to protect their kidneys. A complete list ofcancer drugs can be found on the NCI website.

Anti-angiogenesis is focused on stopping angiogenesis, which is theprocess of making new blood vessels. Because a tumor needs the nutrientsdelivered by blood vessels to grow and spread, the goal ofanti-angiogenesis therapies is to “starve” the tumor. Bevacizumab(Avastin) is an anti-angiogenesis therapy used to treat glioblastomamultiform when prior treatment has not worked.

A remission is when the tumor cannot be detected in the body. Aremission can be temporary or permanent. For most primary brain tumors,despite imaging tests showing that the tumor growth is controlled orthere are no visible signs of a tumor, it is common for a brain tumor torecur.

In glioma, tumor associated microglia/macrophages (TAM) have been shownto participate in tumor growth, tumor invasion, angiogenesis and immunesystem evasion. TAM is subjected to reprogramming in the tumormicroenvironment, leading to an alternate immunosuppressive tumorigenicM2 phenotype. (da Fonseca A C, Badie B. Clin Dev Immunol 2013:264124). Avariety of microglia/macrophage modulating molecules has been shown toswitch the phenotype of TAMs and decrease glioma progression andincrease survival in preclinical studies (El Andaloussi A, et, al. Glia2006; 54:526-35; Hussain S F, et, al. Cancer Res 2007; 67:9630-6;Gabrusiewicz K, et, al. PLoS One 2011; 6:e23902; Markovic D S, et, al.Brain Behav Immun 2011; 25:624-8). Target delivery of immunomodulatorymolecules to TAMs may provide improved efficacy with reduced sideeffects.

Malignant glioma is the most common and most aggressive primary braintumor and despite the advances in treatment, the median survival remainsat 16.4 months. Key challenges faced in the development of effectivetherapies relate to (a) the ability of systemically deliveredchemotherapeutic agents to penetrate the impaired blood brain tumorbarrier (BBTB) and provide homogenous coverage across the entire solidtumor and (b) the ability to target specific cells. Although smallmolecule-based therapeutics can effectively distribute within the tumortissue, they are limited by rapid tumor clearance and off-targetextravasation potentially leading to adverse effects. Recent advances innanotechnology have provided selective tumor accumulation. However, thesize of most nanoparticles limits extravasation and tumor penetration,thus limiting homogeneous solid tumor coverage. Careful tuning ofparticle size and surface charge has been attempted in order to enhancethe nanoparticle distribution profile in subcutaneous tumors.Unfortunately, achieving homogeneous coverage of orthotopic brain tumorshas proven even more challenging. This may be attributed to the lowerpermeability of the BBTB compared to the blood-tumor barrier (BTB) in asubcutaneous tumor, the heterogeneous intervascular spaces and the highinterstitial pressure in brain tumors. Although, some strategies haveattempted nanoparticle delivery through the BBTB via absorptive uptake;passive diffusion through the leaky BBTB fenestrations has only beendemonstrated with molecules smaller than 20 nm and unhindered diffusionthrough the BBTB has been achieved with molecules of 7 nm, thus limitingsystemic administration of most nanoparticle based therapeutics.

It is therefore an object of the present invention to provide animproved method and reagents for delivering drugs to treat brain tumors.

SUMMARY OF THE INVENTION

A composition comprising poly(amidoamine) (PAMAM) hydroxyl-terminateddendrimers covalently linked to or complexed with at least onetherapeutic, prophylactic or diagnostic agent for the treatment oralleviation of one or more symptoms of a brain tumor have beendeveloped. The composition contains one or more ethylene diamine-corepoly(amidoamine) (PAMAM) hydroxyl-terminated generation-4, 5, 6, 7, 8,9, or 10 (G4-10-OH) dendrimers. The G6 dendrimers have demonstratedunexpectedly high uptake, and uniform distribution in to the entirebrain tumor. The dendrimers provide a means for selective deliverythrough the blood brain barrier (“BBB”) of chemotherapeutic,immunotherapeutic and palliative agents. The dendrimers also have theadvantage that multiple therapeutic, prophylactic, and/or diagnosticagents can be delivered with the same dendrimers. In one embodiment, thedendrimers are complexed with or conjugated to two different classes ofcompounds, providing simultaneous delivery. The dendrimers may beadministered alone by intravenous injection, or as part of a multi-prongtherapy with radiation and/or surgery. In one embodiment, the dendrimersare covalently linked to at least one radiosensitizing agent, in anamount effective to suppress or inhibit the activity of DDX3 in theproliferative disease. In another embodiment, the dendrimers arecovalently linked to at least one detectable moiety, in an amounteffective to detect the tumor in the subject. In another embodiment, thedendrimer composition has multiple agents, such as a chemotherapeuticagent, immunotherapeutic agent, an anti-seizure agent, a steroid todecrease swelling, antibiotic, anti-antiogenic agent, and/or adiagnostic agent, complexed with or conjugated to the dendrimers.

The dendrimer composition is preferably administered systemically, mostpreferably via intravenous injection. The composition may beadministered prior to or immediately after surgery, radiation, or both.The composition may be designed for treatment of specific types oftumors, such as gliomas, or through targeting tumors associated withmicroglia/macrophages (TAM).

The examples demonstrated that hydroxyl terminated PAMAM dendrimersdemonstrate unique favorable pharmacokinetic characteristics in aglioblastoma tumor model following systemic administration. Dendrimersrapidly accumulate and are selectively retained in the tumor tissue.This is due at least in part to the small size and near neutral surfacecharge which allow homogeneous distribution of the dendrimer through theentire solid tumor. Dendrimers homogeneously distribute through theextracellular matrix reaching the entire tumor and peritumoral area.Dendrimers intrinsically target neuroinflammation and accumulate in thetumor associated microglia/macrophages (TAMs). Increasing the generationof dendrimers from 4 to 6 can significantly increase dendrimeraccumulation in the tumor without affecting their homogeneousdistribution and targeting of TAMs. The generation 4 and 6 hydroxylterminated PAMAM dendrimers can leak through the blood brain tumorbarrier and selectively accumulate in glioblastoma, not the peritumoralarea, following systemic administration. However, the dendrimers alsoaccumulate in the peritumoral area, thereby having an effect on themigrating front of glioblastoma. These dendrimers intrinsically targettumor associated microglia/macrophages and are retained in these cellsover at least 48 hours. There is no significant accumulation ofdendrimers in the contralateral hemisphere (‘healthy’) where thedendrimers remain in the blood vessel lumen.

Generation 4 (G4) dendrimers rapidly and selectively accumulate and areretained in the tumor tissue despite their rapid clearance from thecirculation. Based on fluorescence quantification and high resolutionfluorescence microscopy dendrimers accumulate over the first 8 hours andare still retained in the tumor at 48 hours. Increasing the generationof dendrimers from 4 to 6 can significantly increase dendrimeraccumulation, AUC and retention in the tumor ˜100-fold without affectingtheir homogeneous distribution and targeting of TAMs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show a synthetic scheme for conjugating a small molecule,BLZ-945, to a G4 hydroxyl-terminated dendrimer.

FIGS. 2A and 2B are graphs illustrating D-Cy5 pharmacokinetics in thebrain (tumor, peritumoral area and contralateral hemisphere) of a rodent9L gliosarcoma model. FIG. 2A is a graph of D-Cy5 concentration in brainareas 15 minutes, 1 hour, 4 hours, 8 hours, 24 hours and 48 hoursfollowing systemic administration. The accumulation is expressed as μgof D-Cy5 per g of tissue. FIG. 2B is a graph of the area under the curve(“AUC”) of D-Cy5. The AUC at 48 hours demonstrates the significantdifference in dendrimer exposure between the tumor and the contralateralhemisphere.

FIG. 3A is a graph of the linear curve fitting of brain quantificationdata. The R² for contralateral, peritumor and tumor are 0.98, 0.99 and0.96 respectively. FIG. 3B shows calculation of permeation constant(Kin) and initial volume of distribution (Vi) in tumor, peritumoral areaand contralateral hemisphere based on brain pharmacokinetics data,Tissue (t)/serum (t) (mL/g) versus AUC serum t/Serum (t) (hr).

FIGS. 4A and 4B depict the characterization of microglia cells(population and activation) in a 9L gliosarcoma inoculated rodent brainusing Imaris software. FIG. 4A is a graph of the image based cell countof the Iba1+ microglia/macrophages population per mm² area in the tumor,ipsilateral hemisphere and contralateral hemisphere. FIG. 4B is a graphof the image based measurement of microglia cell surface to volume ratioas an indication of activation and phagocytic activity ofmicroglia/macrophages in healthy brain, contralateral hemisphere andipsilateral hemisphere of a tumor inoculated brain and tumor tissue. 1+cells D-Cy5 co-localization with Iba1+ TAMs and D-Cy5 co-localizationwith DAPI+ cells. Results are expressed as percent of the total DAPI+cell population. There is no statistical significance between microgliauptake and cell uptake. Statistical *p<0.05; **p<0.001 Statisticalanalysis is based on 3-5 different slices.

FIG. 5 is a graph of the D-Cy5 cell localization analysis 24 hoursfollowing administration using image based measurement of Iba-1+ cellsD-Cy5 co-localization with Iba1+ TAMs and D-Cy5 co-localization withDAPI+ cells. Results are expressed as percent of the total DAPI+ cellpopulation. There is no statistical significance between microgliauptake and cell uptake.

FIGS. 6A-6C are graphs based on the fluorescence based quantification ofD-Cy5 in major organs (brain, liver, lung, spleen, heart, and kidney),serum and urine of 9L gliosarcoma rodent model 24 hours following D-Cy5administration. FIG. 6A is the biodistribution is expressed in percentof injected dose per organ: D-Cy5 accumulation in kidney, urine andother organs. FIG. 6B is a graph of the time dependent concentration ofD-Cy5 in spleen, liver, kidney and serum. Concentration is expressed inpercent of injected dose per g of tissue. FIG. 6C is a graph of thefluorescence based quantification of the plasma pharmacokinetics ofD-Cy5.

FIGS. 7A-7C are graphs of the comparison of accumulation (μg ofdendrimer/g of brain tissue) (FIG. 7A) between G6 and G4 dendrimers in atumor bearing brain as a function of time. The accumulation of G4dendrimers in the tumor/peritumor peaked around 8 hours post injection,and gradually decreased, while G6 dendrimer concentration intumor/peritumor continuously increased. At 48 hours, G6 concentrationwas almost 100 fold higher than G4 dendrimers concentration in thetumor; the area under curve (AUC) plot (FIG. 7B) which demonstrates G6dendrimers have around 100 fold higher brain tumor exposure than G4dendrimers within 48 hours. When the dendrimer concentration in thebrain is normalized by the dendrimer concentration in the serum, thebrain to serum ratio is an indication of the brain targeting ability. G6dendrimers showed higher tumor targeting ability than G4 dendrimers atall the time points (FIG. 7C).

FIGS. 8A and 8B are graphs showing the G4 and G6 dendrimer concentrationin serum and major organs: kidney, liver, spleen as a function of time.FIG. 8A shows that G6 dendrimers showed higher serum concentration andprolonged serum half-life than G4 dendrimers, which contributed to thehigher tumor accumulation and targeting of G6 dendrimers. The dendrimerconcentration was demonstrated as percentage of total injected dose permilliliter of serum. FIG. 8B showed that for G4 dendrimers, kidney hadmost dendrimer accumulation (20%-30%), significantly higher thandendrimer accumulation in liver and spleen (˜0.3%) at different timepoints. For G6 dendrimers, the increase of size greatly decreased therenal filtration and kidney accumulation. The kidney concentration of G6dendrimers was more than 10 fold less than G4 dendrimers (˜1%), andstarted to show clearance from kidney starting from 48 hours. The liveraccumulation of G6 dendrimers was similar to G4 dendrimers, while spleenshowed ˜5 fold higher accumulation, possibly due to the increased uptakeby monocytes.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “therapeutic agent” refers to an agent that can be administeredto prevent or treat one or more symptoms of a disease or disorder.Examples include, but are not limited to, a nucleic acid, a nucleic acidanalog, a small molecule, a peptidomimetic, a protein, peptide,carbohydrate or sugar, lipid, or surfactant, or a combination thereof.

The term “treating” refers to preventing or alleviating one or moresymptoms of a disease, disorder or condition. Treating the disease orcondition includes ameliorating at least one symptom of the particulardisease or condition, even if the underlying pathophysiology is notaffected, such as treating the pain of a subject by administration of ananalgesic agent even though such agent does not treat the cause of thepain.

The phrase “pharmaceutically acceptable” refers to compositions,polymers and other materials and/or dosage forms which are, within thescope of sound medical judgment, suitable for use in contact with thetissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio. The phrase“pharmaceutically acceptable carrier” refers to pharmaceuticallyacceptable materials, compositions or vehicles, such as a liquid orsolid filler, diluent, solvent or encapsulating material involved incarrying or transporting any subject composition, from one organ, orportion of the body, to another organ, or portion of the body. Eachcarrier must be “acceptable” in the sense of being compatible with theother ingredients of a subject composition and not injurious to thepatient.

The phrase “therapeutically effective amount” refers to an amount of thetherapeutic agent that produces some desired effect at a reasonablebenefit/risk ratio applicable to any medical treatment. The effectiveamount may vary depending on such factors as the disease or conditionbeing treated, the particular targeted constructs being administered,the size of the subject, or the severity of the disease or condition.One of ordinary skill in the art may empirically determine the effectiveamount of a particular compound without necessitating undueexperimentation.

II. Formulation

A. Dendrimers

The term “dendrimer” as used herein includes, but is not limited to, amolecular architecture with an interior core, interior layers (or“generations”) of repeating units regularly attached to this initiatorcore, and an exterior surface of terminal groups attached to theoutermost generation. Examples of dendrimers include, but are notlimited to, PAMAM, polyester, polylysine, and PPI. The PAMAM dendrimerscan have carboxylic, amine and hydroxyl terminations and can be anygeneration of dendrimers including, but not limited to, generation 1PAMAM dendrimers, generation 2 PAMAM dendrimers, generation 3 PAMAMdendrimers, generation 4 PAMAM dendrimers, generation 5 PAMAMdendrimers, generation 6 PAMAM dendrimers, generation 7 PAMAMdendrimers, generation 8 PAMAM dendrimers, generation 9 PAMAMdendrimers, or generation 10 PAMAM dendrimers. Dendrimers suitable foruse include, but are not limited to, polyamidoamine (PAMAM),polypropylamine (POPAM), polyethylenimine, polylysine, polyester,iptycene, aliphatic poly(ether), and/or aromatic polyether dendrimers.Each dendrimer of the dendrimer complex may be of similar or differentchemical nature than the other dendrimers (e.g., the first dendrimer mayinclude a PAMAM dendrimer, while the second dendrimer may comprise aPOPAM dendrimer). In some embodiments, the first or second dendrimer mayfurther include an additional agent. The multiarm PEG polymer includes apolyethylene glycol having at least two branches bearing sulfhydryl orthiopyridine terminal groups; however, embodiments disclosed herein arenot limited to this class and PEG polymers bearing other terminal groupssuch as succinimidyl or maleimide terminations can be used. The PEGpolymers in the molecular weight 10 kDa to 80 kDa can be used.

A dendrimer complex includes multiple dendrimers. For example, thedendrimer complex can include a third dendrimer; wherein thethird-dendrimer is complexed with at least one other dendrimer. Further,a third agent can be complexed with the third dendrimer. In anotherembodiment, the first and second dendrimers are each complexed to athird dendrimer, wherein the first and second dendrimers are PAMAMdendrimers and the third dendrimer is a POPAM dendrimer. Additionaldendrimers can be incorporated without departing from the spirit of theinvention. When multiple dendrimers are utilized, multiple agents canalso be incorporated. is not limited by the number of dendrimerscomplexed to one another.

As used herein, the term “PAMAM dendrimer” means poly(amidoamine)dendrimer, which may contain different cores, with amidoamine buildingblocks. The method for making them is known to those of skill in the artand generally, involves a two-step iterative reaction sequence thatproduces concentric shells (generations) of dendritic 0-alanine unitsaround a central initiator core. This PAMAM core-shell architecturegrows linearly in diameter as a function of added shells (generations).Meanwhile, the surface groups amplify exponentially at each generationaccording to dendritic-branching mathematics. They are available ingenerations G0-10 with 5 different core types and 10 functional surfacegroups. The dendrimer-branched polymer may consist of polyamidoamine(PAMAM), polyester, polyether, polylysine, or polyethylene glycol (PEG),polypeptide dendrimers.

In accordance with some embodiments, the PAMAM dendrimers used can begeneration 4 dendrimers, or more, with hydroxyl groups attached to theirfunctional surface groups. The multiarm PEG polymer comprisespolyethylene glycol having 2 and more branches bearing sulfhydryl orthiopyridine terminal groups; however, embodiments are not limited tothis class and PEG polymers bearing other terminal groups such assuccinimidyl or maleimide terminations can be used. The PEG polymers inthe molecular weight 10 kDa to 80 kDa can be used.

In some embodiments, the dendrimers are in nanoparticle form and aredescribed in detail in international patent publication No.WO2009/046446.

Preparation of PAMAM-BLZ-945

As a non-limiting example, below is a synthetic scheme for conjugating asmall molecule, BLZ-945, to a hydroxyl-terminated fourth generationPAMAM dendrimer (PAMAM-OH), using acetic acid, 2-(2-cyclooctyn-1-yloxy)acid and 2-azidoacetic acid as linkers. See FIGS. 1A-1C

Initially, the hydroxyl-terminated fourth generation PAMAM dendrimer(PAMAM-OH) is functionalized into clickable bifunctional dendrimer(intermediate 1) with 9 clickable groups on the surface using2-(2-cyclooctyn-1-yloxy) acid (FIG. 1A). BLZ-945 is reacted with2-azidoacetic acid to form azide-functionalized intermediate via anester bond (FIG. 1B). The resulting azide on the 2-azidoacetyl linker isfurther reacted with the clickable groups of the bifunctional dendrimerto get dendrimer-BLZ-945 conjugate. (FIG. 1C) There are approximatelynine molecules of BLZ-945 conjugated to one molecule of the dendrimer.

The scheme described above is not limited to BLZ-945. Other smallmolecules, for example, a small molecule inhibitor of a SignalTransducer and Activator of Transcription (STAT) protein such as WP1066,and other small molecules such as minocycline and cyclosporine A can beconjugated to the dendrimers as immunomodulatory molecules for TAMstargeting therapies.

B. Coupling Agents and Spacers

Dendrimer complexes can be formed of therapeutically active agents orcompounds (hereinafter “agent”) conjugated or attached to a dendrimer ormultiarm PEG. The attachment can occur via an appropriate spacer thatprovides a disulfide bridge between the agent and the dendrimer. Thedendrimer complexes are capable of rapid release of the agent in vivo bythiol exchange reactions, under the reduced conditions found in body.

The term “spacers” as used herein is intended to include compositionsused for linking a therapeutically active agent to the dendrimer. Thespacer can be either a single chemical entity or two or more chemicalentities linked together to bridge the polymer and the therapeutic agentor imaging agent. The spacers can include any small chemical entity,peptide or polymers having sulfhydryl, thiopyridine, succinimidyl,maleimide, vinylsulfone, and carbonate terminations.

The spacer can be chosen from among a class of compounds terminating insulfhydryl, thiopyridine, succinimidyl, maleimide, vinylsulfone andcarbonate group. The spacer can comprise thiopyridine terminatedcompounds such as dithiodipyridine, N-Succinimidyl3-(2-pyridyldithio)-propionate (SPDP), Succinimidyl6-(3-[2-pyridyldithio]-propionamido)hexanoate LC-SPDP or Sulfo-LC-SPDP.The spacer can also include peptides wherein the peptides are linear orcyclic essentially having sulfhydryl groups such as glutathione,homocysteine, cysteine and its derivatives, arg-gly-asp-cys (RGDC),cyclo(Arg-Gly-Asp-d-Phe-Cys) (c(RGDfC)), cyclo(Arg-Gly-Asp-D-Tyr-Cys),cyclo(Arg-Ala-Asp-d-Tyr-Cys). The spacer can be a mercapto acidderivative such as 3 mercapto propionic acid, mercapto acetic acid, 4mercapto butyric acid, thiolan-2-one, 6 mercaptohexanoic acid, 5mercapto valeric acid and other mercapto derivatives such as 2mercaptoethanol and 2 mercaptoethylamine. The spacer can bethiosalicylic acid and its derivatives,(4-succinimidyloxycarbonyl-methyl-alpha-2-pyridylthio)toluene,(3-[2-pyridithio]propionyl hydrazide, The spacer can have maleimideterminations wherein the spacer comprises polymer or small chemicalentity such as bis-maleimido diethylene glycol and bis-maleimidotriethylene glycol, Bis-Maleimidoethane, bismaleimidohexane. The spacercan comprise vinylsulfone such as 1,6-Hexane-bis-vinylsulfone. Thespacer can comprise thioglycosides such as thioglucose. The spacer canbe reduced proteins such as bovine serum albumin and human serumalbumin, any thiol terminated compound capable of forming disulfidebonds The spacer can include polyethylene glycol having maleimide,succinimidyl and thiol terminations.

The therapeutically active agent, imaging agent, and/or targeting moietycan be either covalently attached or intra-molecularly dispersed orencapsulated. The dendrimer is preferably a PAMAM dendrimer up togeneration 10, having carboxylic, hydroxyl, or amine terminations. ThePEG polymer is a star shaped polymer having 2 or more arms and amolecular weight of 10 kDa to 80 kDa. The PEG polymer has sulfhydryl,thiopyridine, succinimidyl, or maleimide terminations. The dendrimer islinked to the targeting moiety, imaging agents, and/or therapeuticagents via a spacer ending in disulfide, ester or amide bonds.

C. Therapeutic, Prophylactic and Diagnostic Agents

The term “dendrimer complexes” as used herein refers to the dendrimerconjugated to or complexed with one or more therapeutic, prophylactic,or diagnostic agent. The dendrimer complex, when administered by i.v.injection, can preferentially cross the blood brain barrier (BBB) onlyunder diseased condition and not under normal conditions. Preferably theagent(s) is attached or conjugated to PAMAM dendrimers or multiarm PEG,which are capable of preferentially releasing the drug intracellularlyunder the reduced conditions found in vivo. The dendrimer complexeslinked to an agent can be used to perform several functions includingtargeting, localization at a diseased site, releasing the drug, andimaging purposes. The dendrimer complexes can be tagged with or withouttargeting moieties such that a disulfide bond between the dendrimer andthe agent or imaging agent is formed via a spacer or linker molecule.

Representative therapeutic (including prodrugs), prophylactic ordiagnostic agents can be peptides, proteins, carbohydrates, nucleotidesor oligonucleotides, small molecules, or combinations thereof.Representative oligonucleotides include siRNAs, microRNAs, DNA, and RNA.

The term “chemotherapeutic agent” generally includes pharmaceutically ortherapeutically active compounds that work by interfering with DNAsynthesis or function in cancer cells. Based on their chemical action ata cellular level, chemotherapeutic agents can be classified ascell-cycle specific agents (effective during certain phases of cellcycle) and cell-cycle nonspecific agents (effective during all phases ofcell cycle). Examples of chemotherapeutic agents include alkylatingagents, angiogenesis inhibitors, modulators of tumor immune response,aromatase inhibitors, antimetabolites, anthracyclines, antitumorantibiotics, platinum compounds, topoisomerase inhibitors, radioactiveisotopes, radiosensitizing agents, checkpoint inhibitors, PD1inhibitors, APRKinase inhibitors, plant alkaloids, glycolytic inhibitorsand prodrugs thereof.

Representative chemotherapeutics commonly used in treating brain tumorsinclude taxols such as paclitaxel, BCNU, camptothecin, doxycycline,cisplatin, and derivatives, analogues and prodrugs thereof.

Examples of PD-1 inhibitors include, for example, MDX-1106 is agenetically engineered, fully human immunoglobulin G4 (IgG4) monoclonalantibody specific for human PD-1, and pembrolizumab, recently approvedby the US FDA.

Therapeutic agents can include agents which enhance the effect of adifferent therapy, such as radiation. As used herein, the term “aradiation dose sensitizer” means any agent, which when contacted with acell, population of cell or tissue, increases the susceptibility of thatcell, population of cell or tissue to ionizing radiation. In someembodiments, the radiosensitizer is a DDX3 inhibitor, such as thecompound RK-33, or a salt, solvate, stereoisomer, or derivative thereof.

Therapeutic agents include agents which alleviate one or more symptomsof the brain tumor. For example, agents which reduce swelling associatedwith the tumor may be delivered via the dendrimers. Examples includeanti-inflammatory agents such as steroids, for example, methylprednisone, dexamethasone, and fluocinolone acetonide, non-steroidalanti-inflammatory agents such as COX-2 inhibitors, gold compoundanti-inflammatory agents, immunosuppressive agents, salicylateanti-inflammatory agents, ranibizumab, minocycline, and rapamycin. Otheranti-inflammatory drugs include nonsteroidal drug such as indomethacin,aspirin, acetaminophen, diclofenac sodium and ibuprofen.

A peptide drug can be any sequence that is active on TAMs or cancercells. Examples include Peptides (M2pep with the sequence YEQDPWGVKWWYand scM2pep with the sequence WEDYQWPVYKGW) with a Lys3Gly3Ser linkerand a C-terminal biotin tag were purchased from Elim Biopharmaceuticalsat >95% purity. KLA materials were synthesized and purified at >95%purity as follows: M2pepKLA (YEQDPWGVKWWYGGGS-D[KLAKLAK]2), scM2pepKLA(WEDYQWPVYKGWSGGGS-D[KLAKLAK]2), and KLA (D[KLAKLAK]2).

Examples for immunotherapeutic agents targeting TAMS can include colonystimulating factor-1 (CSF-1) receptor inhibitor such as BLZ-945 andPLX3397, MAPKinase inhibitors such as PD98059, a small moleculeinhibitor of STAT (e.g. WP1066), Minocycline, and cyclosporine A.

Other exemplary therapeutic agents include vasodilators andanti-infective agents. Antibiotics include beta-lactams such aspenicillin and ampicillin, cephalosporins such as cefuroxime, cefaclor,cephalexin, cephydroxil, cepfodoxime and proxetil, tetracyclineantibiotics such as doxycycline and minocycline, microlide antibioticssuch as azithromycin, erythromycin, rapamycin and clarithromycin,fluoroquinolones such as ciprofloxacin, enrofloxacin, ofloxacin,gatifloxacin, levofloxacin and norfloxacin, tobramycin, colistin, oraztreonam as well as antibiotics which are known to possessanti-inflammatory activity, such as erythromycin, azithromycin, orclarithromycin. Other agents having activity as anti-excitotoxic agentssuch as valproic acid, D-aminophosphonovalerate,D-aminophosphonoheptanoate, inhibitors of glutamate formation/release,such as baclofen, and NMDA receptor antagonists can also beadministered.

In some embodiments, the molecules can include antibodies, for example,daclizumab, bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®),basiliximab, ranibizumab, and pegaptanib sodium or peptides like SN50,and antagonists of NF.

Exemplary diagnostic agents include paramagnetic molecules, fluorescentcompounds, magnetic molecules, and radionuclides, x-ray imaging agents,and contrast media. These may also be ligands or antibodies which arelabelled with the foregoing or bind to labelled ligands or antibodieswhich are detectable by methods known to those skilled in the art.

Exemplary diagnostic agents include dyes, fluorescent dyes, Nearinfra-red dyes, SPECT imaging agents, PET imaging agents andradioisotopes. Representative dyes include carbocyanine,indocarbocyanine, oxacarbocyanine, thiicarbocyanine and merocyanine,polymethine, coumarine, rhodamine, xanthene, fluorescein,boron-dipyrromethane (BODIPY), Cy5, Cy5.5, Cy7, VivoTag-680,VivoTag-S680, VivoTag-S750, AlexaFluor660, AlexaFluor680, AlexaFluor700,AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780,DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor750, IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, andADS832WS.

Representative SPECT or PET imaging agents include chelators such asdi-ethylene tri-amine penta-acetic acid (DTPA),1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid (DOTA),di-amine dithiols, activated mercaptoacetyl-glycyl-glycyl-gylcine(MAG3), and hydrazidonicotinamide (HYNIC).

Representative isotopes include Tc-94m, Tc-99m, In-111, Ga-67, Ga-68,Gd³⁺, Y-86, Y-90, Lu-177, Re-186, Re-188, Cu-64, Cu-67, Co-55, Co-57,F-18, Sc-47, Ac-225, Bi-213, Bi-212, Pb-212, Sm-153, Ho-166, and Dy-i66.

Targeting moieties include folic acid, RGD peptides either linear orcyclic, TAT peptides, LHRH and BH3.

D. Devices and Formulations

The dendrimers can be administered parenterally by subdural,intravenous, intra-amniotic, intraperitoneal, or subcutaneous routes.

The carriers or diluents used herein may be solid carriers or diluentsfor solid formulations, liquid carriers or diluents for liquidformulations, or mixtures thereof.

For liquid formulations, pharmaceutically acceptable carriers may be,for example, aqueous or non-aqueous solutions, suspensions, emulsions oroils. Parenteral vehicles (for subcutaneous, intravenous, intraarterial,or intratissue injection) include, for example, sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's and fixed oils. Examples of non-aqueous solvents are propyleneglycol, polyethylene glycol, and injectable organic esters such as ethyloleate. Aqueous carriers include, for example, water, alcoholic/aqueoussolutions, cyclodextrins, emulsions or suspensions, including saline andbuffered media.

The dendrimers can also be administered in an emulsion, for example,water in oil. Examples of oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil,mineral oil, olive oil, sunflower oil, fish-liver oil, sesame oil,cottonseed oil, corn oil, olive, petrolatum, and mineral. Suitable fattyacids for use in parenteral formulations include, for example, oleicacid, stearic acid, and isostearic acid. Ethyl oleate and isopropylmyristate are examples of suitable fatty acid esters.

Formulations suitable for parenteral administration can includeantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.Intravenous vehicles can include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose. Ingeneral, water, saline, aqueous dextrose and related sugar solutions,and glycols such as propylene glycols or polyethylene glycol arepreferred liquid carriers, particularly for injectable solutions.

Injectable pharmaceutical carriers for injectable compositions arewell-known to those of ordinary skill in the art (see, e.g.,Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Trissel, 15th ed., pages 622-630(2009)).

Formulations for convection enhanced delivery (“CED”) include solutionsof low molecular weight sales and sugars such as mannitol.

III. Methods of Treatment

A. Disorders or Diseases to be Treated

The dendrimer complex composition, including dendrimers linked to one ormore therapeutic, prophylactic and/or diagnostic agents, can selectivelytarget microglia and astrocytes. Effective blood-brain tumor barrier(BBTB) penetration and uniform solid tumor distribution significantlyenhance therapeutic delivery to brain tumors. Hydroxyl-functionalized,generation-4 or 6 poly(amidoamine) (PAMAM) dendrimers, with their smallsize, near neutral surface charge, selectively localize in cellsassociated with neuroinflammation.

As used herein, the term “proliferative disease” includes cancer andother diseases such as benign and malignant neoplasias and hyperplasias.The term cancer, includes cancers of the CNS and brain, including, butnot limited to, gliomas, glioblastoma, gliosarcoma, astrocytoma,oligodendroglioma, ependymoma, meningioma, medulloblastoma, ganglioma,Schwannoma, craniopharyngioma, cordomas and pituitary tumors.

The tumors may also be of a different origin than the brain. Forexample, the tumors may have originated as alveolar rhabdomyosarcoma,bone cancer, breast cancer, cancer of the anus, anal canal, oranorectum, cancer of the eye, cancer of the intrahepatic bile duct,cancer of the joints, cancer of the neck, gallbladder, or pleura, cancerof the nose, nasal cavity, or middle ear, cancer of the oral cavity,cancer of the vulva, colon cancer, esophageal cancer, cervical cancer,gastrointestinal carcinoid tumor. Hodgkin lymphoma, hypopharynx cancer,kidney cancer, larynx cancer, liver cancer, lung cancer, malignantmesothelioma, melanoma, multiple myeloma, nasopharynx cancer,non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneum,omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectalcancer, renal cancer (e.g., renal cell carcinoma (RCC)), small intestinecancer, soft tissue cancer, stomach cancer, testicular cancer, thyroidcancer, ureter cancer, and urinary bladder cancer.

The dendrimers are administered in a dosage dependent on the tumor sizeand type, location, and other treatments, as well as the agents to bedelivered. Typically, an attending physician will decide the dosage ofthe composition with which to treat each individual subject, taking intoconsideration a variety of factors, such as age, body weight, generalhealth, diet, sex, compound to be administered, route of administration,and the severity of the condition being treated. In general the timingand frequency of administration will be adjusted to balance the efficacyof a given treatment or diagnostic schedule with the side-effects of thegiven delivery system. Exemplary dosing frequencies include continuousinfusion, single and multiple administrations such as hourly, daily,weekly, monthly or yearly dosing.

It will be understood by those of ordinary skill that a dosing regimencan be any amount and for any length of time sufficient to treat a braintumor to reduce size, metastasis, or rate of growth, or to alleviate oneor more symptoms such as swelling, pain, or seizures. Physiciansroutinely determine the length and amounts of therapy to beadministered.

B. Adjunct or Combination Therapies

The dendrimer complexes can be administered in combination with one ormore additional therapeutically active agents, which are known to becapable of treating brain tumors or the symptoms associated therewith.

For example, the dendrimers may be administered to the brain viaintravenous administration or during surgery to remove all or a part ofthe tumor. The dendrimers may be used to deliver chemotherapeuticagents, immunotherapeutic agents, agents to enhance adjunct therapy suchas of a subject undergoing radiation therapy, wherein thepoly(amidoamine) (PAMAM) hydroxyl-terminated dendrimers are covalentlylinked to at least one radiosensitizing agent, in an amount effective tosuppress or inhibit the activity of DDX3 in the proliferative disease inthe brain.

It will be understood by those of ordinary skill in the art, that inaddition to chemotherapy, surgical intervention and radiation therapyare also used in treatment of cancers of the CNS. Radiation therapy, asused herein, means administering ionizing radiation to the subject inproximity to the location of the cancer in the subject. In someembodiments, the radiosensitizing agent is administered in 2 or moredoses and subsequently, ionizing radiation is administered to thesubject in proximity to the location of the cancer in the subject. Infurther embodiments, the administration of the radiosensitizing agentfollowed by the ionizing radiation can be repeated for 2 or more cycles.

Typically, the dose of ionizing radiation varies with the size andlocation of the tumor, but is dose is in the range of 0.1 Gy to about 30Gy, preferably in a range of 5 Gy to about 25 Gy.

In some embodiments, the ionizing radiation is in the form ofsterotactic ablative radiotherapy (SABR) or sterotactic body radiationtherapy (SBRT).

C. Imaging and Diagnostics

The dendrimers are also useful in a method for imaging TAM associatedwith a proliferative disease in a subject. The dendrimers are linked toat least one detectable moiety, administered to the subjectintravenously in an amount effective to detect the TAM in the subject.

The dendrimer compositions can be formulated for theranostic purposes.In other words, the dendrimer compositions can comprise multiplecompositions which include at least one biologically active agent and atleast one detectable moiety. In some cases the at least one biologicallyactive agent and at least one detectable moiety can be the samemolecular entity. Thus, the dendrimer compositions can be used to detectthe proliferative disease or tumor in the body of the subject anddeliver a biologically active agent to the tumor or TAM simultaneously.

The present invention will be further understood by reference to thefollowing non-limiting examples.

Example 1: Administration of Fluorescently Labeled Dendrimer to Gliomasin Rats

Materials and Methods.

The following agents were purchased: hydroxyl terminatedethylenediamino-core PAMAM dendrimer (referred to as dendrimerthroughout, unless otherwise specified) (Dendritech, Midland, Mich.),Methanol (HPLC grade), DMF (HPLC grade), stainless steel beads (FisherScientific, Waltham, Mass.); and Cyanine 5 (Cy5) (GE Healthcare LifeScience, Pittsburgh, Pa.). For confocal microscopy: nuclei counterstain,4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI), Alexa Fluor® 594Goat Anti-Rabbit IgG (H+L) Antibody (Molecular Probes, Eugene, Oreg.);Fluorescent mounting media (Dako, Santa Clara, Calif.); Anti-Iba1,Rabbit (Wako, Osaka, Japan); Lectin from Bandeiraea simplicifolia(BSI-B4) (Sigma-Aldrich, St. Louis, Mo.); Anti-GFAP 488 (eBioscience,San Diego, Calif.); Fluorescein isothiocyanate-dextran (FITC-dextran),average molecular weight 70,000 (Sigma Aldrich, St. Louis, Mo.).

Synthesis of Dendrimer Cy5 (D-Cy5) Conjugates

D-Cy5 was prepared through two steps following the method of MolPharmaceutics, 10:4560 (2013). Briefly, hydroxyl-terminated PAMAMdendrimer was surface-modified with amine groups to make a bifunctionaldendrimer. 6-(Fmoc-amino)caproic acid was used to produce aFmoc-protected bifunctional dendrimer intermediate that was eventuallyde-protected by re-dissolving in piperidine/DMF mixture. Cy5 dye withN-hydroxysuccinimide monoester was reacted with amine groups on thesurface of bifunctional dendrimer. The ‘crude’ products were furtherextensively purified by dialysis. The final D-Cy5 conjugate wascharacterized using ¹H NMR, high-performance liquid chromatography(HPLC) and gel permeation chromatography (GPC). The conjugate was storedas a solid powder at −20° C. and reconstituted at 10 mg/ml with sterile0.9% NaCl on the day of administration.

Tumor Inoculation.

Female Fischer 344 rats, weighing 125-175 g each (Harlan Bioproducts,Indiana, Ind.), were housed in standard facilities and given free accessto food and water. 9L gliosarcoma intracranial implantation wasperformed as described in Neurosurgery 2010, 66, 530-7; J. Neurosurg.2010, 113, 210-7. Briefly, the 9L gliosarcoma (obtained from the BrainTumor Research Center, UCSF, San Francisco, Calif.) was maintained inthe flank of F344. Tumor was surgically excised from the flank of thecarrier animal, sectioned into 1 mm³ pieces and placed in sterile 0.9%NaCl on ice for intracranial implantation. Rats were anesthetized and amidline scalp incision was made to identify the sagittal and coronalsutures. A burr hole was made 3 mm lateral to the sagittal suture and 5mm posterior to the coronal suture. The dura was incised, and using asurgical microscope and gentle suction a small cortical area wasresected. A tumor piece was placed in the resection cavity and the skinwas closed using surgical staples. All animals were treated inaccordance with the policies and guidelines of the Johns HopkinsUniversity Animal Care and Use Committee.

D-Cy5 Administration for Quantification and Immunofluorescence.

For tail vein injections, animals were immobilized and their tails wereheated to induce vasodilation. 3 mg/300 μl of the dendrimer-Cy5 solutionwas administered per animal. For imaging of dendrimer and dextrandistribution, 3 animals were co-injected with a 0.9% NaCl solution of 2mg D-Cy5 and 2 mg dextran-FITC in 300 μl.

To study the dynamics of dendrimer accumulation in the tumor brain,D-Cy5 was injected into 27 tumor inoculated rats when the average tumorsize was 6 mm in diameter and then animals were sacrificed at fixed timepoints (15 minutes, 1 hour, 4 hours, 8 hours, 24 hours, and 48 hours).Magnetic resonance imaging was used to measure intracranial tumor size.Blood was drawn through cardiac puncture and immediately centrifuged tocollect plasma. Brains were harvested and flash frozen on dry ice forfluorescence spectroscopy based quantification or placed in 4% formalinsolution for immunofluorescence.

To study the dendrimer cell uptake, D-Cy5 injection was performed in 3tumor inoculated rats and 3 healthy rats, and animals were sacrificed 24hours after the injection. Brains were harvested and placed in 4%formalin for immunofluorescence study.

To study the pharmacokinetics and biodistribution of dendrimer in plasmaand systemic organs D-Cy5 was injected into 15 tumor-inoculated ratswhich were placed in metabolic cages for urine collection and animalswere subsequently euthanized at fixed time points (15 minutes, 1 hour, 4hours, 8 hours, 24 hours, and 48 hours). Organs were harvested and flashfrozen on dry ice for fluorescence spectroscopy-based quantification orplaced in 4% formalin for immunofluorescence.

Fluorescence Spectroscopy

Fluorescence-based quantification of D-Cy5 conjugates followed theprotocol in Lesniak, Mol Pharm. 2013 Dec. 2; 10(12):4560-71. Briefly,100-150 mg of frozen tissue was homogenized in 1 ml of methanol using ahomogenizer (TissueLyser LT, Giagen) in 2 ml DNA LoBind Eppendorf tubesand subsequently sonicated. Suspensions were diluted to 100 mg/ml andcentrifuged at 15,000 rpm for 15 minutes at 4° C. The resultingsupernatants were subjected to fluorescence spectroscopy. Importantly,prior studies showed that D-Cy5 was stable in plasma, and could berecovered from the tissue intact, without appreciable release of theconjugated Cy5.

For the brain tissue, precise dissection of the tumor was performed andthe peritumoral area was defined as up to 1 mm away from the tumordissection plane. In the contralateral hemisphere 100 mg of thecaudate/putamen with the surrounding white matter area was dissected andused for analysis. For plasma and urine samples a sample of 100 μl ofplasma and urine was mixed with 900 μl of phosphate buffer (0.1M) andanalyzed by fluorescence spectroscopy.

Fluorescence spectra of D-Cy5 conjugates and that obtained from tissueextracts were recorded using a Shimadzu RF-5301 Spectrofluorophotometer(Kyoto, Japan). D-Cy5 calibration curves were constructed, followingevery experiment, under different slit widths using the maximum emissionwavelength of 662 nm after recording spectra from 650 nm to 720 nm withexcitation wavelength of 645 nm. The D-Cy5 concentration was measured inmethanol or phosphate buffer (0.1 M) in solutions ranging from 1 ng/mlto 100 μg/ml. The slit width was chosen based on the observedfluorescence level of different sample sets. For biological samples withlow levels of D-Cy5 (i.e. brain, lung, heart), the excitation andemission slit width was set at 10; for biological samples with highlevels of D-Cy5, (i.e. urine and kidney) excitation and emission slitwidth was set at 3. For the remaining biological samples, excitationslit width of 5 and emission slit width of 10 were used. All calibrationcurves exhibited linearity with R² 0.99. Fluorescence registered fromtissue of non D-Cy5 injected healthy and tumor inoculated rats wassubtracted from the values observed from samples of D-Cy5 injectedtissue in order to account for tissue autofluorescence.

Concentration of D-Cy5 conjugate in the brain was expressed in μg per gof tissue. The concentration of D-Cy5 conjugate in the other organs wasexpressed in percentage (%) of injected dose per g of tissue or % ofinjected dose per organ. Concentrations of the D-Cy5 conjugate in urineand blood were expressed in % of injected dose per ml or % of injecteddose in total amount of urine or plasma. Total plasma concentration wascalculated based on the weight of the animal (J. Nucl. Med., 1985, 26,72-6). The brain and plasma quantification data were analyzed tocalculate the area under the curve (AUC) and the brain to serum ratio.

The permeation constant (K_(in)) and the initial volume of distribution(V_(i)) were calculated in the brain tumor (Nanomedicine (Lond) 2013, 9,111-21; J. Cereb. Blood Flow Metab. 1983, 3, 8-32). The brain to serumratio and the area under the curve of the serum (AUC_((serum))(t)) toserum concentration (Serum(t)) were calculated for each time point andlinear regression analysis was performed in order to get the K_(in) andV_(i) based the following equation:

$\frac{{Brain}(t)}{{Serum}(t)} = {{K_{in}\frac{{AUC}_{serum}(t)}{{Serum}(t)}} + V_{i}}$

For all three regions in the brain, the multiple time-point regressionanalysis showed good linearity with R² equal to 0.98, 0.99, and 0.96 forthe contralateral hemisphere, the peritumoral, and the tumor region,respectively (FIG. 3A).

Immunofluorescence

Freshly harvested tissues were fixed in 4% formalin for 24 hours,followed by a gradient of sucrose solutions before cryosection. Tissueswere then sectioned transversely into 30 μm-thick slices using a LeicaCM 1905 cryostat. Slices were stained with DAPI (nuclei), rabbitanti-Iba1 antibody for microglia/macrophages, and goat anti-rabbit 595secondary antibody. Some slices were stained with isolectin forendothelial cell staining. Slices were then imaged using a confocal LSM710 microscope (Carl Zeiss; Hertfordshire, UK) under 5×, 20×, 40× and63× magnifications. For each slice of tumor-inoculated brains, imageswhere acquired for the tumor, tumor border and contralateral hemisphere.For control (non-tumor) brains, 1-3 representative images were acquired.Settings were optimized to avoid background fluorescence based onnon-injected control rat brains. Laser power, pinhole, gain, offset anddigital gain were selected separately for each magnification and keptconstant throughout the entire study.

Software

For image processing Zen software was used, any adjustments inbrightness and contrast were kept constant throughout the samemagnification images. No adjustments were done on the Cy5 channel.Imaris software was used for cell counting, co-localization andmicroglia surface to volume ratio measurements. Microsoft Excel 2010 andKaleidaGraph 4.0 were used for all calculations, curve fitting andfigure plotting related to the pharmacokinetic study.

Cell Count and Co-Localization

For microglia/macrophage cell count 20× 13×13 tile scan images wereanalyzed and 3-5 slices were analyzed per region. The function ‘spots’was used to identify Iba1+ microglia/macrophages. A diameter thresholdof 4.15 μm was set to eliminate the objects smaller than microglia cellsand an intensity threshold of 26.801 based on ‘Quality’ analysis was setto eliminate the background signal.

To study co-localization 40×, 4×4 tile scan images were used and 3-5slides were analyzed per region. The function ‘spots’ was used toidentify DAPI+ nuclei, Iba1+ microglia/macrophages and D-Cy5+ cells. Forcells with D-Cy5 uptake, the spots with D-Cy5 and DAPI co-localizationwere counted; for microglia cells with D-Cy5 uptake, the spots withDAPI, anti-Iba1 and D-Cy5 co-localization were counted. Estimateddiameters were applied to eliminate the spots with size smaller thancells, and signal thresholds were applied based on ‘Quality’ analysis.The function co-localize spots was used by counting the spots whereD-Cy5 signal and cell signal are within 10 μm next to each other.

For surface to volume ratio analysis of the microglia cells, 3Drepresentation of microglia morphology was acquired in confocalmicroscope using 40× magnification, with 3×3 tile scan, extending 10 μmin the z direction in z-stack. The function ‘surfaces’ was used and theindividual Iba1+ microglia/macrophages were analyzed for surface andvolume of each cell. Tumor area, ipsilateral (non-tumor area),contralateral area, and non-tumor brains were analyzed, approximately150 cells were included for each region. The threshold settings werebased on the diameter of cells (Nat. Neurosci. 2009, 12, 872-8).

Statistical Analysis

Statistical analysis of data was carried out by student's t-test andone-way ANOVA followed by Games-Howell tests with SPSS 18.0 (IBM, Inc.),as needed. Differences were considered statistically significant atp<0.05.

Results

Pharmacokinetics of Systemically Delivered D-Cy5 in Intracranial BrainTumor

FIGS. 2A and 2B show the pharmacokinetic analysis of D-Cy5 in differentregions of tumor bearing rodent brain: tumor, peritumor andcontralateral hemisphere. The AUC at 48 hours was listed separately todemonstrate the large difference between tumor and contralateralhemisphere. Based on fluorescence confocal microscopy, dendrimersrapidly accumulated throughout a 5 mm tumor, as early as fifteen minutesafter systemic administration.

In contrast, in the ‘healthy’ contralateral hemisphere, dendrimersoutlined the blood vessels, and were not observed in the parenchymausing confocal microscopy images of tumor inoculated rodent brainfollowing systemic administration of D-Cy5. Images indicate thehomogeneous distribution of D-Cy5 in the tumor and the restriction ofdendrimer in the blood vessel lumen. Fifteen minutes after systemicadministration, the dendrimer was dispersed throughout the entireintracranial tumor parenchyma. This distribution was not influenced bythe heterogeneity in the tumor parenchyma. There was no appreciablecellular uptake at this time point. At 4 hours post systemic dendrimeradministration, the extracellular distribution in the tumor region haddecreased, which was accompanied by an increased uptake by Iba1+inflammatory cells. The contralateral hemisphere showed relativelyminimal D-Cy5 fluorescence at all the time points.

In order to assess the kinetics of dendrimer accumulation in the brain,a recently developed fluorescence-based semi-quantification method forD-Cy5 was used. The use of the near IR Cy5 wavelength overcomes thetissue autofluorescence challenges. The high sensitivity of this method(0.1 ng/g of tissue) allowed detection of dendrimer accumulation atspecific anatomic locations. In accordance with the confocal microscopyresults, dendrimer rapidly accumulated in the tumor and peritumoralarea, with a peak concentration occurring at 8 hours. See FIG. 3A.Dendrimer gradually cleared from the tumor at a rate of ˜0.01 μg/g/hour,and from the peritumoral area at a rate of ˜0.007 μg/g/hour, reaching aconcentration of 0.2 μg/g of tissue, 48 hours after initial systemicinjection. In the contralateral_hemisphere, the dendrimer accumulationalso peaked at 8 hours, at a concentration ˜8-fold lower than that foundin the tumor area. At 24 hours, traces of dendrimer (0.03 μg/g) could bedetected in the contralateral hemisphere and a ˜14 fold higheraccumulation in the tumor was observed. At 48 hours the AUC was 10 timeshigher in the tumor area in comparison to the contralateral hemisphere,indicating significantly higher (p<0.05) overall exposure of thedendrimer to the tumor. The high and selective retention of dendrimer inthe tumor and peritumoral area was visualized in a low magnificationimage of the tumor stained for astrocytes 24 hours following theadministration of dendrimer.

The vasculature and the amount of BBTB disruption have been demonstratedto differ significantly between the tumor core and a tumor border whichmay play a significant role in drug accumulation. Therefore, bloodvessel endothelial cells were stained in order to examine the differencein vasculature and, therefore, perfusion between the tumor and theperitumoral area. As expected, the peritumoral area and the tumor bordershowed dramatically denser vasculature than the tumor core. However, thedendrimer distribution appeared to be uniform in the tumor.

To further understand the kinetics of dendrimer penetration in the tumorthe permeation constant (K_(in)) and the initial volume of distribution(V_(i)) in the tumor, peritumoral area and contralateral hemisphere,were calculated. See FIG. 3B. Ki. describes the influx of dendrimer fromthe blood to the brain, and was 10 fold higher in the tumor andperitumoral area comparison to the contralateral hemisphere, indicatingthe increased penetration of dendrimers and increased permeability andperfusion of the tumor tissue. V_(i) represents the volume of the braincompartments in rapid equilibrium with plasma, differed significantlybetween the tumor and the peritumoral area indicating a larger volume ofrapid equilibration in the tumor core. In tumor xenografts the hypoxictumor core has increased vascular permeability in comparison to thetumor border and in glioblastoma specifically a distinct difference inthe morphology of the BBTB between the tumor core and the peritumoralarea has been shown, which may contribute to the rapid distribution inthe tumor core as opposed to the peritumoral area.

Bio-Distribution of D-Cy5: Imaging-Based Study in Intracranial BrainTumor

To study the dendrimer distribution in the tumor and peritumoral area,D-Cy5 was co-injected with linear dextran-FITC (70 kDa, ˜6.5 nm radius)which has approximately twice the size of dendrimer. The tumor wasclearly identified based on the increased density of DAPI-positivenuclei. See FIGS. 4A and 4B. At each time point, the dendrimerdistributed homogeneously throughout the whole tumor region and amarkedly decreased signal was observed 24 hours after injection. SeeFIG. 5. In comparison, signal from dextran-FITC was only observed aroundthe tumor border and not in the tumor core, even when the laser powerand gain settings were increased to see the background signal in theFITC channel. Higher magnification images showed that dendrimer rapidlydistributed and delineated the extracellular matrix (ECM) leading to areticular pattern of distribution and gradual accumulation in the cells.On the contrary, dextran showed limited distribution throughout theextracellular matrix but high signal could be seen within the bloodvessel lumen fifteen minutes after injection. At later time points,limited amounts of dextran were retained in the tissue presumably due tolow cellular uptake.

Characterization of Tumor Associated Microglia/Macrophages and CellUptake of Dendrimer

Gliomas produce chemo-attractants and growth factors that promoterecruitment and proliferation of microglia/macrophages. In humanglioblastoma up to 30% of cells can be tumor associated macrophages. Themicroglial distribution was determined in different anatomic locationsof the 9L tumor model, which suggested that the concentration of TAM inthis tumor model is similar to that seen in human glioblastoma. Themicroglia population per mm² was 9-fold higher within the tumor ascompared to the contralateral hemisphere and 2.5-fold higher in healthybrain tissue of the ipsilateral hemisphere as compared to thecontralateral

TAM is reprogrammed in the tumor microenvironment, leading to analternate immunosuppressive M2 phenotype. However, a number of studieshave suggested sustained phagocytic activity of TAM in glioma. Thephagocytic activity of TAM has been suggested to play a key role innanoparticle uptake. When microglia/macrophages change from a resting toan activated form, their morphology is modified from ramified toamoeboid indicative of their increased phagocytic activity. In order toassess the morphology of TAM in the 9L tumor model the surface to volumeratio of the immune cell population (Iba1+) in different anatomicallocations was characterized. The surface to volume ratio (StoV ratio) isconsidered a ‘measure’ of microglial activation. The results indicatethat the microglia/macrophages within the tumor and surrounding thetumor had a significantly (p<0.001) lower StoV ratio in comparison tothe immune cells in the contralateral hemisphere, and in a healthybrain. The mean StoV ratio for tumor associated microglia was lower than1, indicative of their amoeboid state and phagocytic activity. Imagebased cell count of the Iba1+ microglia/macrophages population per mm²area in the tumor, ipsilateral hemisphere and contralateral hemisphere.Image based measurement of microglia cell surface to volume ratio as anindication of activation and phagocytic activity ofmicroglia/macrophages in healthy brain, contralateral hemisphere andipsilateral hemisphere of a tumor inoculated brain and tumor tissue. 1+cells D-Cy5 co-localization with Iba1+ TAMs and D-Cy5 co-localizationwith DAPI+ cells. Results are expressed as percent of the total DAPI+cell population. There is no statistical significance between microgliauptake and cell uptake. Statistical *p<0.05; **p<0.001 Statisticalanalysis is based on 3-5 different slices. See FIGS. 4A and 4B.

The localization of D-Cy5 in the Iba1+ microglia/macrophages cells wasthen calculated. At 24 hours post systemic administration, dendrimerslocalized in the Iba1+ TAM. Iba1+ microglia/macrophages were calculatedto comprise 38% of the total tumor cell population (FIG. 5).Co-localization indicated that approximately half of the TAM populationtook up dendrimers and that the total population of dendrimer-positivecells did not differ quantitatively from the population ofdendrimer-positive Iba1+ microglia/macrophages. Therefore, dendrimerswere taken up almost exclusively by tumor associated macrophages withinthe tumor tissue, while other cells within the tumor region did not havemeasurable dendrimer uptake. In the tumor border (1 mm from the tumoredge based on DAPI stain) the dendrimer-positive microglia/macrophageswere substantially reduced, reflecting the difference in the biologicalprocesses between the tumor core and the tumor border. Dendrimer was notpresent in the ipsilateral non-tumor region or in the contralateralhemisphere.

D-Cy5 cell localization analysis 24 hours following administration wasperformed using image based measurement of Iba-1+ cells D-Cy5co-localization with Iba1+ TAMs and D-Cy5 co-localization with DAPI+cells. Results are expressed as percent of the total DAPI+ cellpopulation. There is no statistical significance between microgliauptake and cell uptake. High magnification (40×) fluorescence confocalimaging of different anatomic locations of a 9L gliosarcoma inoculatedbrain.

Systemic Biodistribution of D-Cy5

After 24 hours systemic administration, fluorescence-quantification ofextracted D-Cy5, suggested that 56% of the dendrimer was excretedthrough the urine, while 32% remained in the kidneys. This correlateswell with the low serum levels (0.66%, FIG. 6A) at this same time point.After 24 hours systemic administration, only 2.5% of the dendrimers wereaccumulated in other major organs. 1.5% of these dendrimers wereretained in the liver and spleen representing the elimination by thereticuloendothelial system (RES). Trace amounts accumulated in thebrain, lung, and heart.

To better understand the kinetics, dendrimer concentration in the serumand major organs was measured over time (FIG. 6B). The kidney had anincreasing accumulation at all chosen time points up to 24 hours. After48 hours from systemic administration, the amount of dendrimer in thekidneys began to decrease. Dendrimer serum level decreased rapidly withonly 4% of the injected dose observed 15 minutes after systemicinjection (FIG. 8A).

FIG. 6A shows the fluorescence based quantification of D-Cy5 in majororgans (brain, liver, lung, spleen, heart, and kidney), serum and urineof 9L gliosarcoma rodent model 24 hours following D-Cy5 administration.The biodistribution is expressed in percent of injected dose per organ;(inserted panel): D-Cy5 accumulation in kidney, urine and other organs.(6B) Time dependent concentration of D-Cy5 in spleen, liver, kidney andserum. Concentration is expressed in percent of injected dose per g oftissue. (57C) Fluorescence based quantification of the plasmapharmacokinetics of D-Cy5.

Renal Accumulation and Distribution

The high concentration of dendrimer in the renal system led to the studyof the distribution in the kidney in order to assess the sites ofaccumulation. Based on fluorescence microscopy, the dendrimeraccumulated in the renal cortex. Dendrimer co-localized with anti-GFAPantibody which stained the peritubular fibroblasts. No presence ofdendrimer was observed in the glomeruli. There was no significantdifference in terms of the clearance through the kidneys among healthyand tumor-bearing rats.

Summary and Conclusions

Malignant glioma is the most common primary brain tumor and results inmore years of life lost than any other tumor. Numerous traditional smallmolecule chemotherapeutic drugs in preclinical and clinical trials failto have a drastic impact in the natural history of the disease due totheir low accumulation and rapid clearance from the tumor followingsystemic administration.

At 48 hours the dendrimer accumulation in the brain tumor is 11 foldhigher than that in the contralateral hemisphere. This ratio stands outin comparison to other non-targeted nanoparticles and is comparable toactively targeted magnetic nanoparticles. Importantly, high retention isalso observed in the peritumoral area. Glioblastoma cells are highlyinfiltrative and can be found in anatomic locations with intact bloodbrain barrier; thus achieving high retention at the peritumoral area isof distinct importance for the design of an effective therapeuticvehicle. Selective retention of dendrimers in the tumor and peritumoralarea in combination with low circulation half-life allows for specificdelivery of chemotherapeutics with limited off target effects.

Example 2: Hydroxyl Terminated Generation 6 PAMAM Dendrimers asTherapeutic Vehicles for the Treatment of Glioblastoma

Materials and Methods

The materials and methods are as described in Example 1, with theexception that both generation 4 and 6 hydroxyl terminated PAMAMdendrimers were utilized.

To study the dynamics of dendrimer accumulation in the tumor brain,dendrimers were injected into tumor inoculated rats when the averagetumor size was 6 mm in diameter and then animals were sacrificed atdifferent time points.

Results

The efficacy of chemotherapeutics is directly associated with the amountof accumulation in the tumor. Increasing the dendrimer size fromgeneration 4 to generation 6 increased their hydrodynamic diameter from˜4.3 nm to ˜6.7 nm without significantly influencing their ζ-potential(Table 1). Hydrodynamic diameter (size) and surface charge (ζ-potential)were measured using dynamic light scattering in PBS, pH 7.4 at roomtemperature. Molecular weight was provided by the supplier.

TABLE 1 Physiochemical properties of hydroxyl terminated dendrimers withgeneration 4 (G4-OH) and generation 6 (G6-OH). Dendrimer MW (kDa) Size +SEM (nm) ζ-potential + SEM (mV) G4-OH 14.1 4.3 ± 0.2 +4.5 ± 0.1 G6-OH58.0 6.7 ± 0.1 0.25 ± 0.4

Example 1 shows that G4 dendrimers selectively accumulate in TAMs whenadministered systemically in 9L gliosarcoma inoculated rats at 24 hrpost D-Cy5 administration. However, the increase of size allowed G6dendrimers to avoid the rapid clearance caused by renal filtration andto circulate longer in the blood, allowing a better localization andretention. G6 dendrimers showed higher serum concentration and prolongedserum half-life than G4 dendrimers, which contributed to the highertumor accumulation and targeting of G6 dendrimers.

FIGS. 7A-7C are graphs of the comparison of accumulation (μg ofdendrimer/g of brain tissue) (FIG. 7A) between G6 and G4 dendrimers in atumor bearing brain as a function of time. The accumulation of G4dendrimers in the tumor/peritumor peaked around 8 hours post injection,and gradually decreased, while G6 dendrimers concentration intumor/peritumor continuously increased. At 48 hours, G6 concentrationwas almost 100-fold higher than G4 dendrimers concentration in thetumor; the area under curve (AUC) plot (FIG. 7B) which demonstrates G6dendrimers have around 100 fold higher brain tumor exposure than G6dendrimers within 48 hours.

When the dendrimer concentration in the brain is normalized by thedendrimer concentration in the serum, the brain to serum ratio is anindication of the brain targeting ability. G6 dendrimers showed highertumor targeting ability than G4 dendrimers at all the time points (FIG.6C). The dendrimer concentration was demonstrated as percentage of totalinjected dose per milliliter of serum. For G4 dendrimers, kidney had themost dendrimer accumulation (20%-30%), significantly higher thandendrimer accumulation in liver and spleen (˜0.3%) at different timepoints. For G6 dendrimers, the increase of size greatly decreased therenal filtration and kidney accumulation. The kidney concentration of G6dendrimers was more than 10 fold less than G4 dendrimers (˜1%), andstarted to showed the evidence of clearance from kidney starting from 48hours. The liver accumulation of G6 dendrimers was similar to G4dendrimers, while spleen showed ˜5 fold higher accumulation, possiblydue to the increased uptake by monocytes). The high serum concentrationprovides a driving force for G6 dendrimers to diffuse across theblood-brain tumor barrier and better target tumor while accumulate inthe tumor with 100-fold higher concentration and AUC (at 48 hours)compared with G4 dendrimers (FIG. 7B).

The high frequency of glioblastoma recurrence occurs from the individualcells that survive the aggressive treatment. For this reason it isimportant that any therapeutic vehicle be able to reach every tumorcell. Recent studies in the nanotechnology field have underlined thatefficient distribution of nanoparticles throughout the tumor tissue as aprerequisite for efficacy. Most of these studies revealed thatnanoparticles with a size range between 10-50 nm could uniformlydistribute through the solid tumor, when administered into the tumor.Larger nanoparticles with size of sub-100 nm, are less likely todistribute homogeneously in the whole tumor bed, due to the fibrotictissue, where no blood vessel exist, interweaving in between the nest ofcancer cells forms diffusion barrier against the homogeneousdistribution of molecules. However, the homogeneous distribution ofnanoparticles also requires them to uniformly extravasate fromvasculature without any hindrance. In the case of glioblastoma, thisrequirement further decreased the upper limit for size cutoff to 10 nm,considering the smaller fenestration and pore size in cranial tumorscompared with subcutaneous tumor. Unhindered diffusion through the BBTBhas been achieved with molecules of 7 nm.

The charge density also influences the penetration of nanoparticles. Itis important for nanoparticles to maintain neutral or slightly positivecharge instead of strongly cationic to prevent the electrostaticallyderived binding-site barrier effect. As a result, G4 OH PAMAM dendrimerswith hydrodynamic diameter of 4.3 nm and neutral surface charge canrapidly distribute through the intervascular spaces in 15 mins andhomogeneously cover the entire 5 mm tumor. Increasing the generation toG6 not only retains the dendrimers' ability to homogeneously distributein the brain tumor, but also prevents dendrimers from clear out fromtumor parenchyma rapidly.

G4 and G6 dendrimers were co-injected intravenously into the tumorbearing rats through tail vein. The brain was fixed and cryo-sectionedaxially. See FIGS. 8A and 8B. FIGS. 8A and 8B are graphs showing the G4and G6 dendrimers concentration in serum and major organs: kidney,liver, spleen as a function of time. FIG. 8A shows that G6 dendrimersshowed higher serum concentration and prolonged serum half-life than G4dendrimers, which contributed to the higher tumor accumulation andtargeting of G6 dendrimers. The dendrimer concentration was demonstratedas percentage of total injected dose per milliliter of serum. The liveraccumulation of G6 dendrimers was similar to G4 dendrimers, while spleenshowed ˜5 fold higher accumulation, possibly due to the increased uptakeby monocytes.

G4 dendrimers had a faster excretion rate than G6 dendrimers. FIG. 8Bshowed that for G4 dendrimers, kidney had most dendrimer accumulation(20%-30%), significantly higher than dendrimer accumulation in liver andspleen (˜0.3%) at different time points. For G6 dendrimers, the increaseof size greatly decreased the renal filtration and kidney accumulation.The kidney concentration of G6 dendrimers was more than 10 fold lessthan G4 dendrimers (˜1%), and started to show clearance from kidneystarting from 48 hours. For G4 dendrimer, excretion from the bloodvessel to tumor tissue started immediately (˜15 min) after i.v.injection, followed with clearance from tumor after 8 hours. After 24hours post injection, G4 dendrimers were retained in the cells. For G6dendrimers, the presence of dendrimers in the tumor tissue was notobserved immediately after administration. The concentration of G6dendrimers gradually increased in the whole observation period. At 48hours post administration, G6 dendrimers showed the highestconcentration in the tumor and peritumor area. Their homogeneousdistribution in glioblastoma including the tumor cell migrating frontand their retention in the tumor for at least 48 hours confirms thedesirability if the dendrimers for efficient delivery of therapeutics.

In addition to homogeneous distribution across the tumor bed uponsystemic administration, dendrimers can also accumulate in theperitumoral region, where deep, active perivasal and perineural invasionof glioma cells into normal nervous tissue occurs. Various techniqueshave been applied for the targeting of peritumoral region such as theusing of GFAP specific monoclonal antibodies to modify thenanoparticles' surface property and the using of magnetic field to guidethe accumulation of magnetic nanoparticles For dendrimers, without anymodification or addition of targeting ligands, the area under curve(AUC) in peritumoral region reaches 60% of that in the tumor region,indicating the ability of dendrimers to intrinsically target peritumoralarea.

Finally, dendrimers target neuroinflammatory cells and largely as wellas exclusively localize in TAMs while resting ramified microglia in thenon-tumor region do not take up dendrimers. Dendrimer specificaccumulation in TAMs with low accumulation in the reticuloendothelialsystem and rapid clearance from the circulation provides a uniqueadvantage for the delivery of antiglioma therapies targeting TAMs.

Modifications and variations of the methods and materials describedherein will be apparent to those skilled in the art and are intended tobe encompassed by the claims.

1-32. (canceled)
 33. A composition comprising hydroxyl-terminatedpoly(amidoamine) (PAMAM) dendrimers covalently linked to or complexedwith one or more chemotherapeutic agents consisting of alkylatingagents, angiogenesis inhibitors, modulators of tumor immune response,aromatase inhibitors, antimetabolites, anthracyclines, antitumorantibiotics, platinum compounds, topoisomerase inhibitors, radioactiveisotopes, radiosensitizing agents, checkpoint inhibitors, APRKinaseinhibitors, plant alkaloids, glycolytic inhibitors and prodrugs thereof.34. The composition of claim 33, wherein the one or more modulators oftumor immune response are selected from the group consisting of colonystimulating factor-1 (CSF-1) receptor inhibitors, MAPKinase inhibitors,inhibitors of STAT, and combinations thereof.
 35. The composition ofclaim 34, wherein the CSF-1 receptor inhibitor is selected from thegroup consisting of BLZ-945 and PLX3397.
 36. The composition of claim33, wherein the chemotherapeutic agents are selected from the groupconsisting of paclitaxel, BCNU, camptothecin, doxycycline, cisplatin,and derivatives, analogues, and prodrugs thereof.
 37. The composition ofclaim 33, wherein the chemotherapeutic agents are selected from thegroup consisting of MDX-1106 and pembrolizumab.
 38. The composition ofclaim 33, wherein the chemotherapeutic agents are selected from thegroup consisting of inhibitors of glutamate formation/release and NMDAreceptor antagonists.
 39. The composition of claim 33, wherein thehydroxyl-terminated PAMAM dendrimers are selected from the groupconsisting of generation 4, generation 5, generation 6, generation 7,generation 8, generation 9, and generation 10 PAMAM dendrimers.
 40. Thecomposition of claim 33, wherein the hydroxyl-terminated PAMAMdendrimers are generation 4 or generation 6 PAMAM dendrimers.
 41. Acomposition comprising hydroxyl-terminated poly(amidoamine) (PAMAM)dendrimers covalently linked to or complexed with one or more diagnosticagents selected from the group consisting of paramagnetic molecules,fluorescent compounds, magnetic molecules, radionuclides, x-ray imagingagents, contrast media, fluorescent dyes, Near infra-red dyes, SPECTimaging agents, PET imaging agents, and radioisotopes.
 42. Thecomposition of claim 41, wherein the SPECT or PET imaging agent is achelator selected from the group consisting of di-ethylene tri-aminepenta-acetic acid (DTPA),1,4,7,10-tetra-azacyclododecane-1,4,7,10-tetraacetic acid (DOTA),di-amine dithiols, activated mercaptoacetyl-glycyl-glycyl-gylcine(MAG3), and hydrazidonicotinamide (HYNIC).
 43. The composition of claim41, wherein the radioisotopes are selected from the group consisting ofTc-94m, Tc-99m, In-111, Ga-67, Ga-68, Gd³⁺, Y-86, Y-90, Lu-177, Re-186,Re-188, Cu-64, Cu-67, Co-55, Co-57, F-18, Sc-47, Ac-225, Bi-213, Bi-212,Pb-212, Sm-153, Ho-166, and Dy-166.
 44. A pharmaceutical formulationcomprising the composition of claim 33 and one or more pharmaceuticallyacceptable excipients.
 45. The pharmaceutical formulation of claim 44formulated for enteral or parenteral administration.
 46. Thepharmaceutical formulation of claim 44 formulated for intravenous,intraperitoneal, or subcutaneous administration.